Riconosciamo Melissa Grunlan per utili discussioni sulla formazione di SAM. Questa ricerca è stata sostenuta in parte dalla Fondazione A. Robert Welch (A-1585).

Preparazione dei reagenti Preparare tampone TUBI (80 TUBI mM, 1 mM MgCl 2, 1mM EGTA, portato a pH 6,8 con KOH). Aliquota e glucosio ossidasi negozio, la catalasi e il glucosio a -70 ° C e taxolo a -20 ° C secondo i protocolli esistenti. 1,2 Aliquotare e tubulina conservare a -70 ° C secondo le istruzioni fornite dal fornitore. Modello microelettrodi Au utilizzando la litografia <ol><li> Tagliare wafer di silicio in 1,5 1 cm &#39;substrati cm usando una scriba o taglierino wafer. Pulire i substrati mettendoli in un bicchiere con acetone sufficiente a coprirli e sonicare per 5 minuti. Senza che i substrati ad asciugare, sciacquare in acetone fresco, poi risciacquare immediatamente in isopropanolo e asciugare con azoto. </li><li> Realizzare microelettrodi Au sui substrati pulita utilizzando le normali, positivo-tono fotolitografia o la litografia a fascio elettronico, a seconda delle dimensioni delle caratteristiche desiderate. 3 Nel progettare la geometria del modello, tenere il diametro di ciascun modello elettrodo inferiore a 100 micron per garantire la copertura del PEG SAM (descritto nella sezione successiva). Includono cuscinetti di contatto (~ 300 mm 2) posizionato 300 a 500 mm dal modello con una linea 1 millimetro di collegare il pad con l&#39;elettrodo (Fig. 1). Posizionare i modelli litografica vicino al centro del supporto per consentire lo spazio intorno al modello per il montaggio della camera di flusso. <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="figura 1" /> Figura 1. Geometria del campione. Si prega di <a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. </li><li> Dopo lo sviluppo del modello nel resistere strato, zero una linea nella resistere dal pad di contatto al bordo del substrato con una pinzetta (Fig. 1). Al deposito di metallo, il graffio permetterà il contatto elettrico tra l&#39;elettrodo e il bordo del supporto. Una camera di deposizione, preferibilmente con due fonti, è utilizzato per depositare lo strato di metallo. Gli elettrodi sono formati dalla deposizione di 2 nm di Cr (per adesione) seguito da 6 nm di Au, senza rompere il vuoto. Un cristallo di quarzo microbilancia attrezzata all&#39;interno della camera può essere utilizzato per misurare lo spessore del film durante la deposizione. </li></ol> Preparare la SAM <ol><li> Preriscaldare il forno e in una zona ben ventilata a 75 ° C. Preparare la soluzione silanizzazione con l&#39;aggiunta di 20 ml di toluene in un becher da 250 ml in vetro. Quindi aggiungere 1 mL PEG-silano (5% v / v) al toluene con una pipetta di plastica. Mescolare nel PEG-silano e pipettando su e giù 5 a 10 volte e poi mescolando con la pipetta per 30 secondi. </li><li> Posizionare i campioni fantasia nel bicchiere con il lato rivolto verso l&#39;alto a motivi geometrici, assicurandosi che essi siano completamente immersi nel PEG-silano soluzione toluene e sono distribuiti uniformemente sul fondo del bicchiere. Porre il bicchiere in forno e cuocere 18-21 ore a 75 ° C. Mentre il SAM si sta formando, preparare il controelettrodo (vedi sotto) e assemblare i componenti aggiuntivi necessari per i passaggi rimanenti. </li><li> Dopo 18-21 ore nel forno, il toluene dovrebbe evaporare lasciando i campioni di fantasia in un viscoso PEG-silano residui. Aggiungere 20 mL di toluene al bicchiere e immergere i campioni per 1-2 minuti. Rimuovere i campioni dal bicchiere e sciacquare con toluene, poi isopropanolo, ed infine asciugare con azoto. A questo punto, i campioni devono look pulito e lo strato SAM dovrebbe essere invisibile ad occhio nudo. SAM preparato in questo modo sono utilizzabili fino a due giorni se conservato in luogo fresco e asciutto (25 ° C, umidità moderata). Infruttuoso risultati formazione SAM in un rivestimento nuvoloso o irregolari residui hued sulla superficie. </li></ol> Realizzare il controelettrodo Nella configurazione stessa camera di deposizione utilizzate per la deposizione di microelettrodi, fare un controelettrodo depositando 1 nm di Cr, seguiti da 4 nm di Au su un x 22 50 mm N ° 1 e n ° 0 coprioggetto di vetro. Il controelettrodo dovrebbe essere quasi trasparente con una tinta grigio chiaro o rosa. Conservare il controelettrodo rivestito-side-up in una capsula di Petri per tenerlo pulito. Tubulina polimerizzano in MT <ol><li> Disgelo senza etichetta tubulina e tubulina rodamina e subito si combinano con un rapporto di 1:2 etichettati in modo da non marcata per ottenere brillanti MTS. Mescolare pipettando su e giù parecchie volte. Polimerizzare la tubulina per 20-40 minuti in un bagno d&#39;acqua riscaldata a 37 ° C. La lunghezza del MTs sarà maggiore per i tempi di polimerizzazione più lunghi. Mentre la tubulina è polimerizzazione, preparare una TUBI-taxolo tampone con l&#39;aggiunta di taxolo a 20 mm in una provetta contenente tampone TUBI. Mescolare bene il taxolo pipettando su e giù parecchie volte e vortex, e quindi inserire il tubo nel bagno riscaldato a 37 ° C. Dopo la polimerizzazione, diluire il MT da 1 / 100 nel warm-taxolo PIPES buffer e scudodalla luce. MT preparato in questo modo si allungano nel tempo e bundle, che può essere ridotto di ulteriore diluizione. </li><li> Preparare una miscela di ossigeno scavenging 10x (OS) combinando 30 mL TUBI tampone a freddo (0-4 ° C), 5 ml di 50% 2-mercaptoetanolo, 5 glucosio ossidasi mL, 5 mL catalasi, glucosio e 5 ml. 2 Il glucosio deve essere aggiunto scorso. Mescolare pipettando su e giù per 3-5 volte dopo l&#39;aggiunta di ogni componente. Mettere il composto OS su ghiaccio, ma rimarrà efficace per circa 2 ore. </li><li> Verificare che la polimerizzazione MT ha avuto successo e che le MT sono stabili sotto alcuni minuti di eccitazione di fluorescenza per immagini la MT preparato con la miscela 1x OS. Per l&#39;imaging L&#39;UST preparato in questo modo, assicurarsi di utilizzare una eccitazione / emissione combinazione di filtri adatti per fluorescenza rodamina. Determinare la diluizione di lavoro ottimale per i filamenti singoli di imaging mediante ulteriori diluizioni MTS di 1 / 10 a 1 / 100 con tampone TUBI e imaging con mix 1x OS. </li><li> Preparare una camera di flusso da sandwich una grande (22 x 50 mm) e piccolo (22 x 22 mm) coprioggetto di vetro con due pezzi di nastro biadesivo messo 2-3 mm. Il nastro può essere facilmente tagliata a strisce mettendolo su un vetrino e il taglio con una lama di rasoio. Introdurre un microlitri alcuni di MTS nella cella di flusso utilizzando una micropipetta. Per l&#39;imaging ad alto ingrandimento, obiettivi a lunga distanza di lavoro può essere richiesto per l&#39;imaging della superficie superiore della camera di flusso. La distanza dal coprioggetto / superficie del campione alla superficie superiore della camera di flusso è determinata dallo spessore del nastro e dovrebbe essere 60-100 mm. </li></ol> Modello MT mediante elettroforesi <ol><li> Preparare una camera di elettroforesi mettendo due sottili strisce di nastro biadesivo in tutto il campione fantasia, SAM rivestite in modo che l&#39;elettrodo è tra le strisce con il percorso di collegamento perpendicolari al nastro (Fig. 2). Posizionare il controelettrodo sul nastro biadesivo con la parte metallica tocca il nastro in modo che il bordo del campione sovrasta il controelettrodo di circa 3 mm (Fig. 2). Diverse lunghezze di taglio 15 cm di filo di rame isolato magnete e nastri 1 cm l&#39;isolamento fuori di entrambe le estremità. Collegare un filo alla zona esposta Au del supporto con una piccola goccia di vernice argento avendo cura di evitare il contatto elettrico con il controelettrodo (questo può essere controllato con un multimetro). Collegare un secondo filo al controelettrodo con vernice argento. Fili attaccati con vernice argento può essere ulteriormente assicurata ai campioni con del nastro adesivo. <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="figura 2" /> Figura 2. Celle di assemblaggio di flusso per un microscopio invertito a fluorescenza. Si prega di <a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg">cliccare qui</a> per vedere una versione più grande della figura 2. </li><li> Introdurre MTs alla diluizione desiderata utilizzando tampone TUBI combinato con OS a 1x concentrazione finale. Immagine a 20X di ingrandimento e individuare il motivo sul wafer di silicio prima di applicare una tensione. In alternativa, la camera di flusso può essere sigillati con smalto per evitare il flusso del fluido e la migrazione MT nel piano della camera di flusso. </li><li> Applicare una tensione fino a 1 V e osservare la migrazione di MTS. Tensioni più basse (fino a circa 0,5 V) possono essere utilizzati se cattura reversibile di MTS è desiderato, ma questo richiede di tenuta della camera di flusso per evitare la dispersione laterale. Smalto funziona bene per sigillare le estremità aperte della camera di flusso. A tensioni di sopra di circa 0,7 V, alcuni adesione MT può essere visto sul modello, anche se sarà debole. A tensioni di sopra di circa 0,9 V, la migrazione MT sarà più veloce, e l&#39;adesione sarà più forte. Sopra circa 1,2 V la probabilità di indurre elettrolisi (formazione di bolle di gas) ed anche per distruggere il microelettrodi aumenta in modo significativo. La migrazione può essere rintracciato regolando il piano verticale di messa a fuoco del microscopio. Se eseguita correttamente, l&#39;UST si localizza alle superfici degli elettrodi. </li></ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

La migrazione di MTS è facilmente visualizzabile usando la microscopia a fluorescenza per la loro luminosità e la fluorescenza di fondo basso. Il metodo è generalmente applicabile a patterning biomolecole, in quanto richiede solo che il biomolecole essere indotto a portare un onere netto di regolazione adeguata del pH del buffer. Elettroosmosi è evitato in questa configurazione perché non ci sono superfici di carico, come le pareti di silice utilizzata in elettroforesi capillare. Divers...

N. 1 vetro borosilicato coprioggetto (22 x 50 mm) sono stati acquistati da VWR. Lucido elettronica grado di tipo p &lt;111&gt; wafer di silicio sono stati acquistati da Addison Engineering, Inc. ad elevata purezza vernice argento è stato ottenuto da Struttura Probe, Inc. (Catalogo # 5001 AB). Isolati calibro sottile filo di rame magnete (~ 0,08 mm (0.003) diametro) è disponibile presso le imprese di fornitura elettronica.

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Vi presentiamo una procedura per la formazione di un poli (glicole etilenico) (PEG) trimetossisilano monostrato auto-assemblato (SAM) su un substrato di silicio con microelettrodi d&#39;oro. Il PEG-SAM si forma in un passo unico assembly ed evita biofouling sulle superfici di silicio e oro. La SAM è utilizzato per microelettrodi cappotto fantasia con lo standard, positivo-tono litografia. Utilizzando il microtubulo come esempio, si applica una tensione continua di indurre la migrazione elettroforetica al SAM-elettrodo rivestito in modo reversibile. Una camera di flusso viene utilizzato per l&#39;imaging e la migrazione elettroforetica patterning microtubuli<em&gt; In situ</em&gt; Usando la microscopia in epifluorescenza. Questo metodo è generalmente applicabile a patterning biomolecole, in quanto impiega l&#39;elettroforesi per immobilizzare molecole bersaglio e quindi non richiede specifiche interazioni molecolari. Inoltre, evita i problemi incontrati nel tentativo di modello le molecole SAM direttamente utilizzando tecniche litografiche. La compatibilità con la litografia a fascio elettronico permette a questo metodo da utilizzare per biomolecole modello su scala nanometrica.

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Vi presentiamo una procedura per la formazione di un poli (glicole etilenico) auto-assemblati monostrato (PEG-SAM) su un substrato di silicio con microelettrodi d&#39;oro. Il PEG-SAM è formato in un unico passaggio ed evita biofouling sulle superfici di silicio e oro. Elettroforesi viene quindi utilizzato per biomolecole patterning fino alla scala nanometrica.

Antivegetativa monostrati auto-assemblati su microelettrodi per Biomolecole Patterning

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

antifouling-self-assembled-monolayers-on-microelectrodes-for

Research

JoVE Journal

Biology

11.4K Views.  Texas A&M University (TAMU). Vi presentiamo una procedura per la formazione di un poli (glicole etilenico) auto-assemblati monostrato (PEG-SAM) su un substrato di silicio con microelettrodi d'oro. Il PEG-SAM è formato in un unico passaggio ed evita biofouling sulle superfici di silicio e oro. Elettroforesi viene quindi utilizzato per biomolecole patterning fino alla scala nanometrica.

Video: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Cellule patterning su elettrodi Tin otticamente trasparente di ossido di indio

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Ricerca

Biologia

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Patterning di cellule staminali embrionali che utilizzano il chip Bio flip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Sistema automatico per le misure di fluorescenza singola molecola di superficie, immobilizzato Biomolecole

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

Proteine ​​saggezza: un banco di lavoro per<em&gt; In silico</em&gt;<em&gt; De novo</em&gt; Progettazione di biomolecole

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Internalizzazione e di osservazione di Fluorescent Biomolecole in Living microrganismi via elettroporazione

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Misura e Analisi di extracellulare Acido prodotte per determinare glicolitica Tasso

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

L&#39;isolamento di regioni genomiche specifici e l&#39;identificazione di molecole da Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Quantificazione di site-specific Protein lisina acetilazione e stechiometria Succinylation usando la spettrometria totale di acquisizione dati-indipendente

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Saggio di screening basato su SA-Galactosidase per l'identificazione dei farmaci senoterapici

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...